This invention relates to nucleic acids and encoded polypeptides which interact with Smad proteins. The invention also relates to agents which bind the nucleic acids or polypeptides. The invention further relates to methods of using such nucleic acids and polypeptides in the treatment and/or diagnosis of disease.
Members of the transforming growth factor-xcex2 (TGF-xcex2) family are multifunctional cytokines with elicit a wide range of cellular effects, including growth inhibition, differentiation and apoptosis (Heldin et al., Nature 390:465-471, 1997). The signaling induced by TGF-xcex2 family members are initiated through a heteromeric transmembrane kinase complex that consists of type I and type II receptors. The activated type I receptor induces the phosphorylation of receptor-activated Smads (R-Smads) which heteromerize with Smad4. These complexes translocate from the cytoplasm to the nucleus to direct transcriptional regulation of responsive genes (Heldin et al., 1997).
Recently, Smad6 and Smad7 were isolated, which form a subfamily among the Smads and function to inhibit the intracellular signaling by R-Smad/Smad4 complexes. Smad6 and Smad7 constitutively associate with type I receptor by blocking association and phosphorylation of R-Smads (Hayashi et al., Cell 89:1165-1173, 1997; Imamura et al., Nature 389:622-626, 1997; Nakao et al., Nature 389:631-635, 1997). Smad6 and Smad7 are rapidly induced by members of the TGF-xcex2 family (Afrakhte et al., Biochem. Biophys. Res. Commun. 249:505-511, 1998), suggesting that inhibitory Smads may take part in a negative feedback control mechanism to modulate the signaling induced by members of TGF-xcex2 family.
The central role of Smads and TGF-xcex2 in cellular processes presents a need for additional factors to modulate Smads and TGF-xcex2 interactions with signal transduction pathways.
Using the yeast two hybrid system, proteins that specifically bind with Smad6 and Smad7 have been isolated. The invention provides these isolated Smad associating proteins (SAPs) and fragments of those molecules, as well as agents which bind such polypeptides, including antibodies. The invention also provides nucleic acid molecules encoding SAPs, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The foregoing can be used in the diagnosis or treatment of conditions characterized by the expression of a Smad associating protein, or in the treatment of conditions characterized by the expression of a SAP, or in the treatment of a condition characterized by the expression of a Smad nucleic acid or polypeptide, or by the inadequate or excessive activity of a Smad polypeptide. The invention also provides methods for identifying pharmacological agents useful in the diagnosis or treatment of such conditions. Here, the identification of several SAPs is presented. The SAPs bind to Smad polypeptides including Smad6 and Smad7 and thus are components of TGF-xcex2 superfamily signaling pathways.
According to one aspect of the invention, isolated nucleic acid molecules are provided. The isolated nucleic acid molecules are nucleic acid molecules which hybridize under stringent conditions to a molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:5 and which code for a polypeptide which binds Smad6, or nucleic acid molecules that differ from the foregoing nucleic acid molecules in codon sequence due to the degeneracy of the genetic code, or complements of the foregoing nucleic acid molecules. Preferably the isolated nucleic acid molecule consists of SEQ ID NO:3 or SEQ ID NO:5.
According to another aspect of the invention, isolated nucleic acid molecules are provided which are unique fragments of nucleotides 1-2399 of SEQ ID NO:3 between 12 and 2398 nucleotides in length or of nucleotides 1-855 of SEQ ID NO:5 between 12 and 854 nucleotides in length. Also provided are complements of the foregoing unique fragments provided that the nucleic acid molecule excludes sequences consisting of GenBank accession numbers AF176069, AF293384, AA305358, AI219112, N33797 and AB030502. In certain embodiments, the isolated nucleic acid molecule consists of at least 22, 25, 30, 40, 50, 75 or 100 contiguous nucleotides. In other embodiments, the isolated nucleic acid molecule consists of between 20 and 32 contiguous nucleotides.
According to still another aspect of the invention, expression vectors including any of the foregoing isolated nucleic acid molecules operably linked to a promoter are provided. Also provided are host cells transformed or transfected with the expression vectors, as well as transgenic non-human animals including the expression vectors.
According to yet another aspect of the invention, methods for producing a polypeptide are provided. The methods include culturing the foregoing host cells under conditions which permit the expression of polypeptide. Preferably the methods include isolating the polypeptide.
In another aspect of the invention, isolated polypeptides are provided which are encoded by the foregoing isolated nucleic acid molecules. Preferred isolated polypeptides include molecules comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:6, fragments or functional variants of SEQ ID NO:4, and a fragments or functional variants of SEQ ID NO:6.
According to still another aspect of the invention, isolated polypeptides are provided which include a fragment or functional variant of SEQ ID NO:2. In certain embodiments the fragment of SEQ ID NO:2 consists of amino acids 1-101+234-424, 106-424 or 234-424.
According to yet another aspect of the invention, an isolated complex of polypeptides is provided. The complex includes one of the foregoing polypeptide bound to a polypeptide selected from the group consisting of Smad6, Smad7 and fragments thereof.
Also included as an aspect of the invention are isolated polypeptides which bind selectively a polypeptide encoded by the foregoing isolated nucleic acid molecules, provided that the isolated polypeptide is not a Smad, STAM or cyclin polypeptide. In certain embodiments, the isolated polypeptide binds to an epitope defined by a polypeptide consisting of the sequence of SEQ ID NOs:2, 4 or 6. In other embodiments, the isolated polypeptide is an antibody fragment selected from the group consisting of a Fab fragment, a F(ab)2 fragment or a fragment including a CDR3 region selective for a SAP polypeptide. In still other embodiments the isolated polypeptide is a monoclonal antibody, a humanized antibody or a chimeric antibody.
According to still another aspect of the invention, methods for modulating TGF-xcex2 superfamily signal transduction in a mammalian cell are provided. The methods include contacting the mammalian cell with an amount of an agent which increases the amount of a Smad associating protein selected from the group consisting of SAP1/AMSH (SEQ ID NO:2), SAP2 (SEQ ID NO:4), SAP3 (SEQ ID NO:6), Hsp40 homolog (U40992; SEQ ID NO:8), Uba80 (X63237; SEQ ID NO:10), Tax-1 binding protein (U33822; SEQ ID NO:12), rabaptin-5 (NMxe2x80x94004703; SEQ ID NO:14), and 26S proteinase S5a (U51007; SEQ ID NO:16) or a fragment thereof in the cell effective to reduce TGF-xcex2 superfamily signal transduction in the mammalian cell. In certain embodiments, the agent is a nucleic acid molecule encoding one of the foregoing polypeptides.
According to another aspect of the invention, methods for regulating the cell cycle in a mammalian cell are provided. The methods include contacting the mammalian cell with an amount of an agent which increases the amount of SAP2 (SEQ ID NO:4), or a fragment thereof, in the cell effective to bind a cyclin and regulate the cell cycle in the mammalian cell.
In further aspects of the invention, methods for identifying lead compounds for a pharmacological agent are provided. In certain embodiments, the methods include forming a mixture comprising a Smad6 or Smad7 polypeptide, a SAP polypeptide, and a candidate pharmacological agent, incubating the mixture under conditions which, in the absence of the candidate pharmacological agent, permit a first amount of specific binding of the SAP polypeptide by the Smad6 or Smad7 polypeptide, and detecting a test amount of the specific binding of the SAP polypeptide by the Smad6 or Smad7 polypeptide. A reduction of the test amount of specific binding relative to the first amount of specific binding indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which disrupts the Smad6-SAP or Smad7-SAP binding, and an increase of the test amount of specific binding relative to the first amount of specific binding indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which enhances the Smad6-SAP or Smad7-SAP binding. Preferably the SAP polypeptide is selected from the group consisting of SAP1/AMSH, SAP2, SAP3 and fragments thereof.
In other embodiments, the methods include forming a mixture comprising an ALK kinase, a Smad polypeptide, a SAP polypeptide, and a candidate pharmacological agent, incubating the mixture under conditions which, in the absence of the candidate pharmacological agent, permit a first amount of specific binding of the SAP polypeptide by the Smad polypeptide, and detecting a test amount of the specific binding of the SAP polypeptide by the Smad polypeptide. A reduction of the test amount of specific binding relative to the first amount of specific binding indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which disrupts the Smad-SAP binding, and an increase of the test amount of specific binding relative to the first amount of specific binding indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which enhances the Smad-SAP binding. In preferred embodiments, the SAP polypeptide is selected from the group consisting of SAP1/AMSH, SAP2, SAP3 and fragments thereof, the Smad polypeptide is selected from the group consisting of Smad2, Smad3, Smad4, Smad6, Smad7 and fragments thereof, and the ALK kinase is selected from the group consisting of ALK5, constitutively activated ALK5, ALK6, constitutively activated ALK6 and fragments thereof having kinase activity.
The use of the foregoing compositions in the preparation of a medicament is also contemplated.
These and other aspects of the invention will be described in further detail in connection with the detailed description of the invention.